The quantitative determination of analytes in body fluids is of great importance in the diagnoses and maintenance of certain physiological abnormalities. For example lactate, cholesterol and bilirubin should be monitored in certain individuals. In particular, the determination of glucose in body fluids is of great importance to diabetic individuals who must frequently check the level of glucose in their body fluids as a means of regulating the glucose intake in their diets. While the remainder of the disclosure herein will be directed towards the determination of glucose, it is to be understood that the procedure and apparatus of this invention can be used for the determination of other analytes upon selection of the appropriate enzyme. The ideal diagnostic device for the detection of glucose in fluids must be simple, so as not to require a high degree of technical skill on the part of the technician administering the test. In many cases, these tests are administered by the patient which lends further emphasis to the need for a test which is easy to carry out. Additionally, such a device should be based upon elements which are sufficiently stable to meet situations of prolonged storage.
Methods for determining analyte concentration in fluids can be based on the electrochemical reaction between the analyte and an enzyme specific to the analyte and a mediator which maintains the enzyme in its initial oxidation state. Suitable redox enzymes include oxidases, dehydrogenases, catalase and peroxidase. For example, in the case where glucose is the analyte, the reaction with glucose oxidase and oxygen is represented by equation (A). ##STR1##
In a colorimetric assay, the released hydrogen peroxide, in the presence of a peroxidase, causes a color change in a redox indicator which color change is proportional to the level of glucose in the test fluid. While colorimetric tests can be made semi-quantitative by the use of color charts for comparison of the color change of the redox indicator with the color change obtained using test fluids of known glucose concentration, and can be rendered more highly quantitative by reading the result with a spectrophotometric instrument, the results are generally not as accurate nor are they obtained as quickly as those obtained using a biosensor. As used herein, the term biosensor is intended to refer to an analytical device that responds selectively to analytes in an appropriate sample and converts their concentration into an electrical signal via a combination of a biological recognition signal and a physico-chemical transducer. Aside from its greater accuracy, a biosensor is an instrument which generates an electrical signal directly thereby facilitating a simplified design. In principle, all the biosensor needs to do is measure the time and read the current. Furthermore, a biosensor offers the advantage of low material cost since a thin layer of chemicals is deposited on the electrodes and little material is wasted.
Referring to the above equation (A), a suitable electrode can measure the formation H.sub.2 O.sub.2 due to its introduction of electrons into the test fluid according to equation B: ##STR2## The electron flow is then converted to the electrical signal which directly correlates to the glucose concentration.
In the initial step of the reaction represented by equation (A), glucose present in the test sample converts the oxidized flavin adenine dinucleotide (FAD) center of the enzyme into its reduced form, (FADH.sub.2). Because these redox centers are essentially electrically insulated within the enzyme molecule, direct electron transfer to the surface of a conventional electrode does not occur to any measurable degree in the absence of an unacceptably high cell voltage. An improvement to this system involves the use of a nonphysiological redox coupling between the electrode and the enzyme to shuttle electrons between the (FADH.sub.2) and the electrode. This is represented by the following scheme in which the redox coupler, typically referred to as a mediator, is represented by M: ##STR3##
In the scheme, GO(FAD) represents the oxidized form of glucose oxidase and GO(FADH.sub.2) indicates its reduced form. The mediating species M.sub.OX /M.sub.red shuttles electrons from the reduced enzyme to the electrode thereby oxidizing the enzyme causing its regeneration in situ which, of course, is desirable for reasons of economy. The main purpose for using a mediator is to reduce the working potential of the sensor. An ideal mediator would be reoxidized at the electrode at a low potential under which impurity in the chemical layer and interfering substances in the sample would not be oxidized thereby minimizing interference.
Many compounds are useful as mediators due to their ability to accept electrons from the reduced enzyme and transfer them to the electrode. Among the mediators known to be useful as electron transfer agents in analytical determinations are the substituted benzo- and naphthoquinones disclosed in U.S. Pat. No. 4,746,607; the N-oxides, nitroso compounds, hydroxylamines and oxines specifically disclosed in EP 0 354 441; the flavins, phenazines, phenothiazines, indophenols, substituted 1,4-benzoquinones and indamins disclosed in EP 0 330 517 and the phenazinium/phenoxazinium salts described in U.S. Pat. No. 3,791,988. A comprehensive review of electrochemical mediators of biological redox systems can be found in Analytica Clinica Acta. 140 (1982), Pp 1-18.
Among the more venerable mediators is hexacyanoferrate, also known as ferricyanide, which is discussed by Schlapfer et al in Clinica Chimica Acta., 57 (1974), Pp. 283-289. In U.S. Pat. No. 4,929,545 there is disclosed the use of a soluble ferricyanide compound in combination with a soluble ferric compound in a composition for enzymatically determining an analyte in a sample. Substituting the iron salt of ferricyanide for oxygen in equation (A) provides: ##STR4## since the ferricyanide is reduced to ferrocyanide by its acceptance of electrons from the glucose oxidase enzyme.
Another way of expressing this reaction is by use of the following equation (C): ##STR5## The electrons released are directly equivalent to the amount of glucose in the test fluid and can be related thereto by measurement of the current which is produced through the fluid upon the application of a potential thereto. Oxidation of the ferrocyanide at the anode renews the cycle.
As is apparent from the above description, a necessary attribute of a mediator is the ability to remain in the oxidized state under the conditions present on the electrode surface prior to the use of the sensor. Any reduction of the mediator will increase the background current resulting in the biosensor reading being biased. It has been discovered that these mediators do tend to be reduced over time, especially under conditions of stress, thereby diminishing the usefulness of the sensors to which they are applied.
In published international patent application PCT/US92/01659 there is disclosed the use of potassium dichromate as an oxidizing agent in a colorimetric reagent strip. The purpose of the oxidizing agent is to oxidize impurities in other reagent components to improve the colorimetric sensor's stability. This publication mentions U.S. Ser. No. 07/451,671 (now U.S. Pat. No. 5,288,636) and characterizes it as describing a system in which a reduced mediator is re-oxidized by the application of a potential and measuring the current after a specific time to determine the concentration of the analyte. More specifically, the '636 patent requires the complete oxidation of the glucose by glucose oxidase. As the enzyme is reduced by the glucose, the ferricyanide reacts with enzyme to produce ferrocyanide. The ferrocyanide produced by this enzymatic reaction is combined with ferrocyanide produced during storage. This latter ferrocyanide is the result of a reaction between ferricyanide and impurities found in materials deposited with the glucose oxidase and ferricyanide. The '636 patent makes no distinction between ferrocyanide produced between these two sources.
It would be desirable, and it is an object of the present invention to provide a method whereby the undesired reduction of mediator compounds stored on an electrodes surface can be reversed to minimize its effect on estimating the analyte values in fluid test samples with very low analyte concentrations.
It is a further object to provide such a method in which the accuracy of the analyte determination is enhanced.
It is a further object to provide such a method wherein the analyte is glucose.
An additional object is to provide a mathematical means for further enhancement of the accuracy of the analyte determination.
It is a further object to provide apparatus for accurately determining analyte values.
It is a further object to provide such apparatus that is simple and economical to manufacture.